Lithium sulfide-based materials have been considered as potential positive electrodes for the next generation batteries. Lithium sulfide is the fully lithiated form of sulfur, i.e., they share the same high theoretical capacity. However, it has the benefit of already containing lithium, which allows making cells with lithium-free negative electrodes. Lithium sulfide, however, shares with sulfur the polysulfide dissolution drawback upon cycling. One possible solution to this problem is to envelop the active material particles with carbonaceous materials. In this work, we investigate the effect of a nitrogen-rich carbon coating on lithium sulfide particles. The effect of such coating on the surface properties and electrochemistry of lithium sulfide cathodes is investigated in details, in particular, regarding its interaction with fresh vs. aged electrolyte. The polymerization of dioxalane (DOL) due to aging is found to affect the electrochemistry of lithium sulfide and, interestingly, to improve the cycling performance. Sulfur-based cathodes, however, still possess a multitude of problems that need to be solved. The main challenge posed by sulfur is the high solubility of the intermediates formed during cycling  are finally formed. The first and most obvious consequences of polysulfides dissolution are low Coulombic efficiencies and loss of active material during battery operation.Lithium-sulfur batteries (LSBs) have attracted much attention in recent years, with the prospect of replacing lithium-ion batteries (LIBs) as energy source for automotive and, especially, flight applications as soon as 2030 , often by enveloping the active material's particles with an inert coating, such as carbon . The 1:1 volume ratio mixture of Dimethoxyethane  and 1,3-Dioxolane  was used as the electrolyte solvent. Prior to mixing, the two solvents were individually dried using 3Å molecular sieves (Sigma Aldrich) to reach water contents below 20 ppm as determined via Karl-Fischer titration (Mettler-Toledo Titrator Compact C30). Appropriate amounts of lithium bis(trifluoromethanesulfonyl)imide  and lithium nitrate  were dissolved into the DME-DOL solvent mixture to achieve concentration of 1 and 0.25 mol L−1, respectively, using aluminum bottles. This electrolyte was used soon after its preparation  or after storage . Besides the aging, both the electrolytes were stored in sealed aluminum bottle inside the glove box.Chemical and electrode preparations, infrared measurements, and cell assembly were carried out in a glove box (MBraun) filled with argon (O2S-CC) was prepared using ethylenediamine-containing lithium sulfide (Li2S-En) as precursor. Li2S-En synthesis, carbon coating procedure, and electrode preparation is described in a previous work  with the conductive carbon  and the binder . The respective weight ratios were 53:37:10. The mixed powders were then dispersed with N-Methyl-2-pyrrolidone , and the resulting slurry cast over aluminum current collectors using the doctor blade method. Finally, the electrode tapes were dried at 60 °C under Ar.LiFor the determination of the electrolyte electrochemical stability window, carbon electrodes composed of Super C65 and PVdF were prepared in the 85:15 weight ratio. NMP was added to prepare the slurry, which was cast over the aluminum current collector as well.−1 and kept for 5 h. The resulting carbonized sample (N-rich carbon) was composed of 62% carbon, 33% nitrogen, 2% hydrogen, and 3% oxygen . This composition is rather similar to that of the carbon coating in Li2S-CC. The N-rich carbon was used to prepare electrodes as well, in which Super C-65, N-rich carbon, and PVdF were mixed in a 70:15:15 weight ratio, dispersed in NMP, and cast over aluminum foil, and dried at 120°C under vacuum for 48 h.Carbon electrodes were also prepared using nitrogen-rich carbon derived from carbonization of 1-ethyl-3-methylimidazolium tricyanomethanide . In short, EMImTCM was placed in an alumina boat inside a horizontal tube furnace under argon flow. The temperature was raised to 500°C at a rate of 5°C min2S-CC based cells were activated by charge at a C/20 rate  up to 4 V vs. Li/Li+, followed by cycling at C/10 between 1.9 and 3.0 V vs. Li/Li+.Electrochemical tests were performed using three electrode T-type cells (Swagelok), with lithium metal as both the reference and counter electrodes. The cells were assembled using 100 μL of electrolyte soaked into glass fiber separators (Whatman GF/A). For the capacity retention experiments, the cells stored at 20°C in climatic chamber (Binder) were galvanostatically cycled using a battery tester (Maccor S4000). The LiElectrochemical impedance spectroscopy (EIS) measurements were performed using a BioLogic VMP3 multi-channel potentiostat. For that, the cells were cycled using the same procedures as mentioned above. However, after each discharge, the cell was allowed to rest at open-circuit for 2 h prior to measuring impedance. Spectra were recorded between frequencies of 200 kHz and 100 mHz, using a 5 mV signal amplitude.+. After cycling, each cell was disassembled inside a glove box, and the electrolyte recovered by centrifuging the glass fiber separators at 6,000 rpm for 10 min. The recovered electrolytes were analyzed via ATR-IR.The anodic electrochemical stability of the electrolytes was studied by cyclic voltammetry (CV), where the potential was scanned from 3 to 4 V vs. Li/Li2S-CC-based electrodes when using either the fresh (Li2S-CC_F) or the aged (Li2S-CC_A) electrolyte. It is evident that the electrolyte aging has a large impact on the cell performance. Surprisingly, the aged electrolyte provides higher discharge capacity as well as capacity retention upon 100 cycles. In order to better understand the reason behind this effect, the discharge curves of the cathode in each electrolyte were closely examined. The inset of 2S-based electrode after the initial activation charge leading to fully oxidized S. The first region (I) shows a potential plateau (around 2.4 V vs. Li/Li+) followed by a slopy region, both associated with the reduction of molecular sulfur (S8) to medium chain polysulfides . Afterwards, a second region (II) characterized by a long plateau at lower potential (around 2.1 V vs. Li/Li+) appears, resulting from the reduction of lithium polysulfides to form the final solid discharge products   is formed in each charge, until a minimum is reached. In 2S-CC_A cell delivered a stable capacity upon cycling, the Li2S-CC_F cell showed a lower and more rapidly decreasing capacity in this region. This indicates that understanding the effect of the N-rich carbon-coating and the aged electrolyte on this voltage region is vital to a deeper comprehension of the lithium sulfide electrochemistry.−1 due to the –CH3 deformation in DME . Li+ is known to coordinate the oxygen atoms in ether molecules  was investigated.The spectra of Li2S-CC_F  show a s2S-CC_F . The dep(2S-CC_A  some dif2S-CC electrodes. As seen in +. For the fresh electrolyte, comparable peaks were observed with both kinds of carbon electrodes in the first cycle. The same occurred in the second cycle, where almost no oxidation current was recorded, indicating that the carbon electrodes underwent a passivation process upon the previous scan. However, the recorded peak current was substantially larger with the aged electrolyte, especially for the electrode solely composed by SuperC 65 and the binder.2S, which leads to the improved performance of such electrodes in the aged electrolyte.The electrolyte was then extracted from such cells and analyzed. The infrared spectra of the electrolytes after cycling  show the2S-based cathode was studied. The presence of polyDOL in the aged electrolyte has an unexpected, but appreciable impact on the performance of the Li2S electrodes, improving the performance of the N-rich carbon-coated Li2S electrodes. The obtained results suggest that PolyDOL can form a stable, hydrophilic passivation layer over the N-rich carbon-coating, which improves the lithium sulfide formation during discharge leading to larger discharge capacities.In summary, the effect of N-rich carbon-coating of LiThe datasets generated for this study are available on request to the corresponding author.LL performed the experiments. LL and AV analyzed the experimental data. All authors wrote the manuscript and conceived the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background. Effects of veneering porcelain thickness and background shade on the shade match of zirconia-based restorations are unclear. The aim of this study was to evaluate the impacts of veneering porcelain thickness and background shade on the shade match of zirconia-based restorations.Methods. Forty A2 shade veneered zirconia disk specimens (10 mm in diameter) were fabricated, with veneering porcelain thicknesses of 1.6, 1.8, 2.0 and 2.2 mm. Three backgrounds were made of A2 shade composite resin (A2), nickel-chromium alloy (NC) and amalgam (AM). The veneered zirconia specimens were placed on the backgrounds. CIELab values were measured with a spectrophotometer. ΔE values were measured to determine color differences between the specimens and the A2 VITA classical shade (target shade). ΔE values were compared with an acceptability threshold (ΔE=3.7). Repeated measures ANOVA, Bonferroni, and 1-sample t-test were used to analyze data (P<0.05).Results. Mean ΔE values ranged between 1.9 and 5.0. The veneering porcelain thickness, the background shade and their interaction affected the ΔE (P<0.0001). The minimum veneering porcelain thickness for the shade match was 2 mm for NC and 1.8 mm for AM.Conclusion. Veneering porcelain thickness and background shade affected the shade match of zirconia-based restorations. With dark-shaded backgrounds, the amount of veneering porcelain thickness needed for the shade match might be beyond acceptable clinical limits. Tooth-shaded backgrounds are esthetically advocated rather than dark-shaded backgrounds in zirconia-based restorations. Zirconia copings represent benefits of high fracture strength,4 proper optical properties5 and a white-to-ivory color.6 Veneering porcelains create a tooth-like appearance due to their proper shade, translucency and tooth-like appearance.7 Consequently, zirconia-based restorations have become popular in dentistry.8Zirconia-based restorations are an acceptable treatment option in restorative dentistry because of their appropriate strength and esthetics.*, a* and b* denote lightness, redness-greenness, and yellowness-blueness, respectively.10 Additionally, the CIELab is used to determine the color difference between two objects. A single value, known as ∆E, is calculated from the formula: ΔE*ab= [(L*2- L*1)2+ (a*2- a *1)2+ (b*2- b*1)2]1/2 in order to measure the color difference.10 Thereafter, the ∆E is compared with acceptability and perceptibility thresholds in order to evaluate the visibility of the color difference to human eyes.14 The CIELab system has been reasonably employed to determine the shade reproduction of different ceramics and restorations.18In order to quantify the color of an object, different color systems have been developed in color science. One of the most commonly applied color systems in dentistry is CIELab, in which L10 Depending on the shade, brand and thickness, zirconia ceramics exhibit different absolute translucencies (visible light transmittance percentage) from low (20%) to ultra (49%) levels. 20 Therefore, zirconia might manifest the color of its underlying materials such as backgrounds and luting agents, leading to an improper shade for zirconia-based restorations.9 A 1-mm-thick zirconia ceramic is needed to create an acceptable masking ability regardless of background color.21 The background L* value affects the masking ability of an 0.5-mm-thick white zirconia ceramic.22 A zirconia-based restoration commonly includes a 0.3- to 0.5-mm-thick zirconia coping and a 1-mm-thick veneering porcelain. Although an 0.4-mm-thick zirconia coping alone produces acceptable masking ability on tooth-colored backgrounds, it might be insufficient for masking metal backgrounds.23 Zirconia-based restorations with an 0.4-mm-thick coping might not be color-matched with natural teeth when placed on gold alloy posts and cores.24 An 0.4-mm-thick zirconia coping alone represents a relative masking ability on color backgrounds, which might lead to color mismatches;24 however, when this zirconia coping is layered with an 0.4-mm-thick veneering porcelain, the resultant color might be further adjusted.25 Moreover, the shade of zirconia-based restorations on a dark background might depend on veneering thickness.26 Some color mismatches have been reported at the cervical area of zirconia-based restorations with 0.3 and 0.5-mm-thick copings.27 Controversially, no significant differences have been reported between CIELab values of zirconia-based crowns on metal backgrounds and composite resin backgrounds.28Zirconia ceramics are optically semi-translucent materials.Various backgrounds and veneering porcelain thicknesses might be used in zirconia-based restorations, inducing different color results. However, according to the controversial results derived from the literature, effects of veneering porcelain thickness and background shade on the shade match of zirconia-based restorations are not clearly understood. Therefore, the aim of this in vitro study was to evaluate the effects of veneering porcelain thickness and background shade on the shade match of zirconia-based restorations. The null hypothesis was that the veneering porcelain thickness and background shade would not affect the shade match of zirconia-based restorations.23 Thus, 40 veneered zirconia disk specimens with 0.4 mm zirconia thickness and 4 veneering porcelain thicknesses  were fabricated. The range of veneering porcelain thickness was selected based on a pilot study.A sample size of n=10 was determined in each study group by considering α=0.05, β=0.1, the study design and previous studies.A computer-aided design/computer-aided manufacturing system  was employed to mill zirconia blanks  and to prepare zirconia disks. The zirconia disks were 0.4 mm in thickness and 10 mm in diameter. The zirconia disks were shaded with an A2 shade coloring liquid  through a 2-minute-immersion process. The zirconia disks were sintered at 1480°C through a 12-hour process in a sintering furnace  and were then adjusted to achieve the intended thickness of 0.4±0.02 mm using a zirconia polishing kit . The zirconia disk was eliminated from the study in case of lack of the intended thickness. Finally, the zirconia disks were cleaned in a 98% ethanol solution and air-dried.An A2 shade feldspathic veneering ceramic for zirconia frameworks  was used to veneer zirconia disks with regard to intended thicknesses using manual add-on technique. The veneered zirconia disks were fired for 69 minutes at a heat rate of 55°C/min from 500°C to 910°C, cooled to room temperature, and polished by using a porcelain polishing laboratory kit . A 3-step polishing procedure was performed using green, purple and yellow polishing disks, as instructed by the manufacturer. The veneered zirconia disks were adjusted to achieve the intended thicknesses (±0.02) using the same porcelain polishing/adjusting kit. The veneered zirconia disk was eliminated from the study in case of lack of the intended thickness. The veneered specimens were cleaned and dried with the aforementioned methods.2 to fabricate the composite resin background (A2). A wax pattern was cast to prepare the nickel‒chromium background (NC). The amalgam alloy was triturated and condensed in a plastic mold to fabricate the amalgam background (AM). All the backgrounds were 10 mm in diameter and 10 mm in height  .Three cylindrical backgrounds were made from an A2 shade light-polymerized composite resin , a nickel‒chromium alloy , an amalgam alloy  Yellow Caps, Densply Sirona, York, PA, USA). The composite resin was applied to a plastic mold and incrementally polymerized with a light-polymerizing unit  for 40 seconds with an intensity of 800 mW/cmn height .21,22 CI23 This mold prevented external lights, supplied a seat for the backgrounds and matched the conditions of spectrophotometry for all the specimens.24 The specimens were seated on the backgrounds without an intermediate.25 Then the CIELab color measurements were performed by an expert operator at the center of the specimens 3 times for each specimen and the average values were recorded .30 The spectrophotometer confirmed the A2 shade for the tab. In order to determine the color difference between the specimens and the target shade, ∆E was calculated from this formula: ΔE*ab= [(ΔL*)2+ (Δa*)2+ (Δb*)2]1/2. An acceptability threshold of ΔE=3.711 was assumed to assess the color differences between the specimens and the target shade and to judge the specimens’ shade match.The same spectrophotometer and a customized putty mold were employed for color measurments.recorded . Also thData were analyzed with SPSS 21 . Kolmogorov-Smirnov test indicated the normal distribution of data in all the studied groups (P>0.05). The effects of veneering porcelain thickness, background shade and their interaction on the CIELab and ∆E values were evaluated using repeated-measures ANOVA. Pairwise comparisons of the studied groups were performed using the Bonferroni correction. The ΔE values of the studied groups were compared with the threshold for acceptability (ΔE=3.7) with STATA  using one-sample t-test. The 0.05 level of significance was considered for all the tests.The mean CIELab and ΔE values for the veneering porcelain thicknesses of 1.6, 1.8, 2.0, and 2.2 mm for the studied backgrounds  are presented in One-sample t-test was used to compare the means of ΔE values with the acceptability threshold (ΔE=3.7). The null hypothesis of ΔE≤3.7 was rejected for NC in 1.6 (P<0.0001) and 1.8 (P<0.0001), and for AM in 1.6 (P<0.0001), while it was not rejected for NC in 2.0 (P=1) and 2.2 (P=1), for AM in 1.8 (P=0.744), 2.0 (P=1), and 2.2 (P=1), and for A2 in all thickness groups (P=1).According to the results of this study, which indicated significant differences in the CIELab and ΔE values in relation to veneering porcelain thickness and background shade, the null hypothesis of the study was rejected. With 0.4-mm zirconia coping, the minimum porcelain thickness for the shade match was 2 mm for NC and 1.8 for AM, while all the tested thicknesses resulted in shade match for A2.9 Veneering porcelains are more translucent than zirconia.9 Thus, a background might express its color under the zirconia coping, affecting the resultant color of a zirconia-based ceramic. The resultant color is the outcome of the ceramic and background colors. Since the backgrounds exhibited different CIELab values, they affected the resultant color in different degrees. The background color initiates the difference in the minimum veneering porcelain thickness needed for esthetics. A greater color difference between the background and the target shade needed a greater veneering porcelain thickness to gain a proper shade match. This is why the general ranking for the proper veneering porcelain thickness needed for the backgrounds was NC>AM>A2  on metal backgrounds and composite resin backgrounds. Their result on the background’s effect was consistent with the present study. Both studies showed that increasing the total restoration thickness beyond the clinically recommended restoration thickness (1.5 mm) led to the color match in zirconia-based restorations regardless of the background color.Suputtamongkol et al23 assessed the effect of zirconia coping thickness and background type on the color masking ability of zirconia-based restorations. They reported that the thickness of zirconia coping should be at least 0.8 mm in order to attain an ideal masking ability on nickel-chromium alloy, while it could be 0.4 mm for tooth-colored backgrounds. However, the present study remarked the veneering ceramic thickness for shade matching. Since the translucency of feldspathic ceramics is more than that of zirconia ceramics, greater thickness for feldspathic ceramics than for zirconia ceramics is needed to achieve an ideal masking ability. However, feldspathic ceramics are optically more similar to tooth structures of dentin and enamel than zirconia ceramics.9 Therefore, the shade/translucency matching of zirconia-based restorations seems more achievable with increasing the veneering ceramic thickness than with increasing the zirconia coping thickness. However, both studies confirmed an increase in the restoration thickness with dark-shaded backgrounds for esthetics.Tabatabaian et al24 reported that background shade , total ceramic thickness (1 and 1.5 mm), and zirconia coping brand  affected the resultant color of zirconia-based restorations with 0.4-mm-thick zirconia coping layered with IPS e.max Press Ceram. They showed that only Lava crowns on gold alloy cores could not create a color match, because their ∆E color difference values were more than the acceptability threshold (close to 5.5). The discrepancies in the results of the studies on the effects of ceramic thickness and background shade might be attributed to the difference in zirconia coping and veneering ceramic brands, control groups, and acceptability thresholds used by the studies.Oh and Kim32 Therefore, with consideration of both esthetic and mechanical properties, the use of tooth-shaded backgrounds rather than dark metal backgrounds is recommended for zirconia-based restorations. An increase in zirconia coping thickness might be a solution for the esthetics in zirconia-based restorations, which was not evaluated in this study. The assessment of the effect of zirconia coping thickness on the color match of zirconia-based restorations is suggested for future research studies.According to the results of this study, increasing the veneering porcelain thickness of zirconia-based restorations improves the esthetic outcomes; however, this might increase the risk of porcelain chipping.9 Among these factors, veneering ceramic thickness and background shade were investigated in this study and accordingly, using a background with a shade corresponding to the target shade is advised instead of dark-shaded backgrounds  for zirconia-based restorations. In case of a dark-shaded background, increasing the restoration thickness might be considered. However, an increase in zirconia coping thickness might be more advantageous than an increase in veneering porcelain thickness alone regarding both mechanical and esthetic properties.The color of zirconia-based restorations might be affected by factors such as background, luting agent, zirconia coping , veneering ceramic , glaze, surface staining, and laboratory procedures .This study did not evaluate some affective factors such as luting agent, thickness and brand of zirconia coping, brand of dentin veneering ceramic, enamel veneering ceramic, glaze, veneering technique, and external staining. Therefore, evaluation of these factors is suggested for future investigations.Within the limitations of this in vitro study, the following conclusions were drawn: Veneering porcelain thickness and background shade affected the shade match of zirconia-based restorations.With dark-shaded backgrounds, the amount of veneering porcelain thickness needed for the shade match might be beyond the acceptable clinical limits.In order to create the shade match for zirconia-based restorations, tooth-shaded backgrounds are advocated rather than dark-shaded backgrounds.The authors declare no conflict(s) of interest related to the publication of this work.FT contributed to the concept and design of the work. The acquisition, analysis, and interpretation of data were accomplished by FT, KA, and MN. FT, KA, and MN drafted and revised the manuscript critically for intellectual content. All the authors read and approved the final manuscript.This study was supported by a grant from the Research Deputy of School of Dentistry of Shahid Beheshti University of Medical Sciences, Tehran, Iran [grant number 11325].The authors thank the Research Deputy of School of Dentistry of Shahid Beheshti University of Medical Sciences for the financial support (the grant No. 11325), Mehr Dental Lab for their CAD/CAM support, and Mr Aliakbar Deghost for his laboratory work in preparing specimens.Not applicable.


Biomaterials that have been newly implanted inside the body are the substratum targets for a “race for the surface”, in which bacterial cells compete against eukaryotic cells for the opportunity to colonize the surface. A victory by the former often results in biomaterial-associated infections, which can be a serious threat to patient health and can undermine the function and performance of the implant. Moreover, bacteria can often have a ‘head start’ if implant contamination has taken place either prior to or during the surgery. Current prevention and treatment strategies often rely on systemic antibiotic therapies, which are becoming increasingly ineffective due to a growing prevalence of antibiotic-resistant bacteria. Nanostructured surfaces that kill bacteria by physically rupturing bacterial cells upon contact have recently emerged as a promising solution for the mitigation of bacterial colonization of implants. Furthermore, these nanoscale features have been shown to enhance the adhesion and proliferation of eukaryotic cells, which is a key to, for example, the successful osseointegration of load-bearing titanium implants. The bactericidal activity and biocompatibility of such nanostructured surfaces are often, however, examined separately, and it is not clear to what extent bacterial cell-surface interactions would affect the subsequent outcomes of host-cell attachment and osseointegration processes. In this study, we investigated the ability of bactericidal nanostructured titanium surfaces to support the attachment and growth of osteoblast-like MG-63 human osteosarcoma cells, despite them having been pre-infected with pathogenic bacteria. MG-63 is a commonly used osteoblastic model to study bone cell viability, adhesion, and proliferation on the surfaces of load-bearing biomaterials, such as titanium. The nanostructured titanium surfaces used here were observed to kill the pathogenic bacteria, whilst simultaneously enhancing the growth of MG-63 cells in vitro when compared to that occurring on sterile, flat titanium surfaces. These results provide further evidence in support of nanostructured bactericidal surfaces being used as a strategy to help eukaryotic cells win the “race for the surface” against bacterial cells on implant materials. Bacterial contamination of implanted biomaterials is difficult to prevent or mitigate ,2, and tStaphylococcus aureus cells with similar levels of bactericidal efficiency. In parallel, nanostructured surfaces with increased degrees of surface roughness have been shown to improve adhesion and proliferation of eukaryotic cells, which is a key to successful osseointegration [In the context of bacteria developing increasing resistance to antimicrobials drugs, bactericidal strategies based on physical, rather than chemical, mechanisms are becoming increasingly important ,23. Nanoegration ,27,28. Begration ,30,31. FStaphylococcus aureus and Pseudomonas aeruginosa.Several in vitro and in vivo models have been developed to study the effects and the interaction between pathogenic bacteria and eukaryotic cells during the processes of simultaneous competitive colonization of implant surfaces. For example, in studies by Pérez-Tanoira et al., host cell viability and integration with an implant surface was found to strongly depend on the concentrations of bacteria present, with bacterial colonization being shown to be reduced in the presence of human cell attachment . SimilarCommercially pure (CP) American Society for Testing and Materials (ASTM) grade 2 titanium billets were used to manufacture Ti discs. Potassium hydroxide was purchased from Merck Millipore . Analytical grade ethanol  and acetone  were used to clean the Ti discs. Methicillin and gentamicin were purchased from Sigma-Aldrich .d = 10 mm) were cut into discs with thickness of 2 mm, using a dicing machine . The discs were then polished, using silicon carbide grinding paper with a grit size of P1200. Prior to hydrothermal etching, Ti discs were further cleaned, using ultrasonic agitation in MilliQ water, 100% ethanol, 100% acetone, and 50% ethanol, in that order, for 6–8 min per cycle. The cleaned Ti discs were then allowed to dry at 37 °C for 24 h. The polished and cleaned Ti disks (referred to as AR-Ti) were used as control, non-modified planar titanium surfaces.CP grade 2 Ti billets  following the protocol reported elsewhere , except Staphylococcus aureus CIP 65.8T, obtained from the Culture Collection of the Institute Pasteur , and Pseudomonas aeruginosa ATCC 9721 obtained from the American Type Culture Collection . Bacterial stocks were prepared in a 20% glycerol nutrient broth  and stored at −80 °C. Fresh bacterial suspensions were prepared for each of the strains at the infective dose of 105 cells per mL for S. aureus and 103 cells per mL for P. aeruginosa in phosphate buffered saline (PBS) . AR-Ti and HTE-Ti disks were immersed into bacterial suspensions and incubated for 6 h at 37 °C in dark conditions. All experiments were conducted with at least three independent technical replicates. Bacterial strains used in this study were methicillin- and gentamicin-susceptible Dynamic bactericidal efficacy was assessed using a standard plate count method following the protocol described elsewhere . PathogeConfocal laser scanning microscopy (CLSM) was used to visualize the proportion of viable and non-viable bacteria on titanium surfaces. A 1 mL aliquot of bacterial suspension of each bacterial strain was placed on the surface of cleaned and sterilized AR-Ti and HTE-Ti discs and allowed to incubate for 18 h at 25 °C. After incubation, the discs were gently washed with copious amounts of sterile MilliQ water to remove non-attached cells following a previously developed experimental protocol . Five sa® 9 and propidium iodide fluorescent dyes. The SYTO® 9 dye permeates viable and non-viable cells, binding to nucleic acids and fluorescing green when excited by a 485 nm wavelength laser. Propidium iodide is only capable of entering cells that have undergone significant membrane damage (and are therefore considered to be nonviable), binding with higher affinity to the nucleic acids present in the bacterial cells than the SYTO® 9 dye and fluorescing red when excited by a 535 nm wavelength laser. Images were recorded using a Fluoview FV3000 microscope  at 60× magnification with water immersed lens. Images were taken across ten different fields of view to get a representation of the entire sample.The bacteria were stained using a LIVE/DEAD BacLight Bacterial Viability Kit, L7012 , which contained a mixture of SYTOThe mammalian cell lines were stained with a LIVE/DEAD Viability/Cytotoxicity Kit  composed of calcein AM and ethidium bromide with the excitation and emission waves of 494/517 nm and 528/617 nm, respectively. Prior to the assay, the samples were first washed with PBS and stained with the dye mixture for 15 min. Then, the samples were washed again with PBS before the assay.High-resolution electron micrographs were obtained using a field emission SEM  at 3 kV under 15,000× magnification. To assess cellular morphology of bacterial and MG-63 cells, titanium discs were sputter-coated with gold, using a Dynavac CS300  prior to imaging.2 for every independent experiment. All the following assessments were performed after 1, 3, and 5 days of seeding when 100% confluency was reached, and three independent technical replicates were performed for each type of surface.The MG-63 cell line was obtained from Sigma-Aldrich (USA) and was cultured in Dulbecco’s Modified Eagle’s medium , supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Sigma-Aldrich). All laboratory experiments were approved by the Biosafety Project 2014/SBC01. Cells were grown to reach 80% confluency and then were trypsinized using 0.25% trypsin/EDTA . Cells were seeded on pre-infected titanium surfaces at a density of 10,000 cells/cmTo perform immunohistochemistry staining, cells were gently washed with PBS, fixed in 4% p-formaldehyde for 15 min, permeabilized in 0.1% Triton X for 5 min, then blocked with 1% bovine serum albumin (BSA) for 60 min. Image-IT® FX Signal Enhancer (Invitrogen) was also used during fixation to enhance fluorescent staining. Fixed cells were treated with primary antibody overnight, followed by secondary antibody. Actin filaments were visualized by staining the cells with Alexa Fluor 488 conjugated Phalloidin (Invitrogen). Nucleus were labeled using TO-PRO3 (Invitrogen). Samples with stained cells were then placed in a glass-bottomed disc for imaging.To perform the 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, the medium was removed, and the cells were washed with PBS solution. Then a fresh medium containing 10% of MTS solution  was added to the samples and they were incubated at 37 °C for 1.5 h. After the incubation period, the medium was transferred to a 96-well plate  and the absorbance was recorded at 490 nm using Polarstar Omega microplate reader .ca. 25 °C) in the dark. LDH stop buffer solution was added at the end of the incubation and the absorbance was recorded at 490 nm.The supernatant of the medium was transferred to 96-well plate  and LDH reaction mix (Promega) was added to the supernatant. Then the mixture was incubated for 30 min at room temperature (ca. 25 °C) for 15 min. After the incubation, the supernatant was then transferred to the 96-well plate  with the added BCA (Sigma-Aldrich) working solution, prepared according to manufacturer’s instructions, and incubated at 37 °C for 30 min. The absorbance was recorded at 562 nm.The medium was removed from the samples and the samples were washed with PBS once. Then mammalian protein extraction agent was added to the samples to extract the proteins out from the cells and incubated at room temperature , according to the manufacturer’s protocol. After RNA extraction, the samples were treated with DNase to eliminate any genomic DNA, and the remaining RNA was then converted to DNA. Prior to quantifying the samples, the quality of DNA was measured using Nanodrop . Each RT-PCR was performed with the following reagents: 10 μL of 2× SensiFAST SYBR & Fluorescein Mix (Bioline), 200 ng of DNA in the samples, 1 μL of each forward and reverse primer (10 μM) and an appropriate volume of nuclease-free water to bring the total reaction volume to 20 μL. The reactions were prepared in 96-well plates sealed with optical quality sealing tape . The reactions were analyzed using MyiQTM single-color detection system  with the following cycling conditions: polymerase activation at 95 °C for 1 min, followed by 40 cycles of denaturation (95 °C for 15 s), primer annealing (54 °C for 30 s) and extension (72 °C for 15 s). Data were collected during the annealing step. A melt curve was obtained by increasing the set-point temperature from 60 °C to 95 °C with a step of 0.5 °C every 10 s. GAPDH was used as a housekeeping control. Differential gene expression fold change was calculated using 2−ΔΔCT (Livak) method [T was calculated from the equation below, taking samples of MG-63 cells grown on AR-Ti as a calibrator. With the equation below, ΔCT values are provided from real-time PCR instrumentation where it is the difference between the target gene and the reference gene (in this work is GAPDH).qRT-PCR was used to quantify the levels of gene expression for collagen type 1 (Col 1), alkaline phosphatase osteocalcin (ALP), and osteopontin (OPN), with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene to normalize the expression level. The primer sequences of each gene were obtained from Marinucci et al . To prepS. aureus CIP 65.8T or P. aeruginosa ATCC 9721 for 6 h at their respective infective doses [HTE-Ti surfaces have previously been shown to possess high bactericidal activity against pathogenic bacteria and even antibiotic-resistant strains ,30,31,34ve doses ,44. An ive doses . The subThe results of examination of pre-infected titanium surfaces after 6 h indicated that both species of pathogenic bacteria were successfully inactivated as confirmed through direct plate count technique , while vP. aeruginosa cells within a few minutes of direct contact [P. aeruginosa cells is seen to be greater than the killing rate against S. aureus cells. Within 3 h of incubation, most P. aeruginosa cells were inactivated by HTE-Ti surfaces, however, it took significantly longer to kill the majority of the S. aureus cells. Although S. aureus cells have greater rigidity than the P. aeruginosa cells, both bacterial strains experienced mechanical stress due to direct contact with the nanostructures that led to the rupturing of the cell membranes at the end of the 6 h incubation period, as shown in The mechanism by which a nanostructured surface, such as that of HTE-Ti, kills bacteria is based on the physical rupturing of the bacterial cell membrane, which experiences mechanical stress as the cells attach onto the nanostructured surfaces ,25. This contact . While t contact . This inP. aeruginosa cells on the AR-Ti surfaces from day 1. Comparatively, the MG-63 cells were viable and could coexist with the S. aureus cells on the AR-Ti surface until day 3. Although the MG-63 cells were viable on the AR-Ti surfaces until day 3, many of these cells still retained their spherical shape. In contrast, on the HTE-Ti surfaces, the MG-63 cells were able to elongate to form their bipolar morphology from day 1.The subsequent colonization of MG-63 cells on pre-infected titanium surfaces showed that these eukaryotic cells were able to attach and proliferate on the pre-infected HTE-Ti surfaces to 100% confluency over 5 days. The MG-63 cells were able to attach onto both the HTE-Ti and AR-Ti surfaces on day 1 . Cells oP. aeruginosa cells as the bacterial cells were able to grow, starving the osteoblast-like cells from the nutrients, killing the MG-63 cells on day 1, as a result. In contrast, the MG-63 cells were able to attach and grow over the AR-Ti surface infected with S. aureus cells until day 3. Eventually, the bacteria overgrew the surface, resulting in the eventual death of the MG-63 cells on day 5. P. aeruginosa cells are known to be more virulent than S. aureus cells, as they tend to secrete toxins when invading host cells [The morphology, attachment, and viability of the MG-63 cells on the pre-infected surfaces were studied after 1, 3, and 5 days of incubation . The seest cells ,48. HencP. aeruginosa or S. aureus cells were able to express vinculin and actin, which are proteins known to be responsible for promoting cell attachment to surfaces. The images presented in P. aeruginosa and S. aureus bacteria, respectively. Similar protein expression was demonstrated by MG-63 cells grown on sterile AR-Ti surfaces, which served as a control (Immunohistochemistry staining of vinculin, actin, and TO-PRO3 were performed on the osteoblast-like cells in order to visualize the expression of the proteins responsible for their attachment and growth, and to observe how these changed over the course of a 5-day incubation period. The MG-63 cells on the HTE-Ti surfaces pre-infected with either  control .P. aeruginosa and S. aureus cells, respectively. The results of the LDH assay show that there is a decrease in LDH concentration from day 1 to day 5 of incubation of the MG-63 cells grown on both the pre-infected HTE-Ti and sterile AR-Ti surfaces, whereas a rapid increase of cell density and protein concentration on the MG-63 cells was observed on both surfaces. The cell density on the pre-infected HTE-Ti surface increased significantly compared to that observed for the cells on the AR-Ti surface on day 3. Additionally, the cells on the HTE-Ti surface possessed a slightly higher protein concentration on day 1 compared to that of the cells on the AR-Ti surface on day 1. Even though the HTE-Ti surfaces were infected with either S. aureus or P. aeruginosa cells, the bioassay results pertaining to the MG-63 cells exhibited no significant difference between these groups, as can be seen by the data presented in Various bioassays, such as LDH, MTS cell proliferation, and BCA, were also used to further assess the wellbeing of the cells on the pre-infected surfaces. The data presented in The osteogenesis-related gene expression of the MG-63 cells was monitored using qRT-PCR to investigate the response of the MG-63 cells to the unique surface features present on the bactericidal HTE-Ti substratum. The gene expression quantification results presented in The results of the immunohistochemistry staining presented in S. aureus or P. aeruginosa cells. This provides further evidence that the HTE-Ti surfaces were able to specifically target the killing of pathogenic bacteria, whilst simultaneously allowing the MG-63 cells to grow and proliferate over the surface in a similar manner to that observed on the non-structured, smooth Ti surfaces. With an ageing population continuing to increase the demand for prosthetics and implants, prevention of implant-associated bacterial infections, especially from multidrug-resistant bacteria, is increasingly becoming a major international healthcare challenge. Given that nanostructured HTE-Ti has been shown to selectively eliminate bacterial contamination without the need for any antimicrobial agents, this bactericidal strategy represents a significant potential for application in the medical implant industry, as well as in many other areas where bacterial contamination is of significant concern, such as in the food processing and handling industry.The results obtained in this study provide evidence that the nanostructured HTE-Ti surfaces exhibited not only bactericidal properties but also displayed biocompatibility towards MG-63 cell attachment, proliferation, and differentiation. There was no significant difference observed in the bioassay and gene expression results for the MG-63 cells grown on the HTE-Ti surfaces that had been pre-infected with


Staphylococcus aureus (MRSA) with slight hemolytic and cytotoxic activities on eukaryotic cells. In this study, the effects of Cath-A on Acinetobacter baumannii, and Pseudomonas aeruginosa isolated from medical instruments were studied. Cath-A inhibited the growth of bacterial cells in the range of 8–16 μg/mL and 16-≥256 μg/mL for A. baumannii and P. aeruginosa, respectively. The peptide significantly removed the established biofilms. To display a representative approach for the cost-effective constructions of peptides, the recombinant Cath-A was cloned in the expression vector pET-32a(+) and transformed to Escherichia coli BL21. The peptide was expressed with a thioredoxin (Trx) sequence in optimum conditions. The recombinant peptide was purified with a Ni2+ affinity chromatography and the mature peptide was released after removing the Trx fusion protein with enterokinase. The final concentration of the partially purified peptide was 17.6 mg/L of a bacterial culture which exhibited antimicrobial activities. The current expression and purification method displayed a fast and effective system to finally produce active Cath-A for further in-vitro study usage.The emergence of antimicrobial resistance among pathogenic microorganisms has been led to an urgent need for antibiotic alternatives. Antimicrobial peptides (AMPs) have been introduced as promising therapeutic agents because of their remarkable potentials. A new modified cathelicidin-BF peptide (Cath-A) with 34 amino acid sequences, represents the potential antimicrobial effects against methicillin-resistant  Acinetobacter baumannii and Pseudomonas aeruginosa are among the common causes of morbidity and mortality in hospitals [Microbial drug resistance is considered a global public health problem. The spreading of multidrug-resistant (MDR) bacteria threatens the healthcare system. The increasing cost of treatment, prolonged hospitalization, and failure to prevent serious infections are affected by the overuse of antibiotics ,2. Becauospitals ,6,7.Acinetobacter and Pseudomonas spp. in nosocomial infections all around the world [Acinetobacter and Pseudomonas spp. from Iran [Acinetobacter and Pseudomonas spp. to attach and form biofilms on both biotic and abiotic surfaces is a critical role to their pathogenesis in hospitalized patients. Their ability to form MDR microbial biofilms causes a varied range of infections such as skin, wound, and urinary tract infections, to septicemia in immune-suppressed or immune-compromised patients [These non-fermenting Gram-negative pathogens are quickly resistant to almost all antibiotic classes such as β-lactam, aminoglycosides, and quinolones agents because of their improved intrinsic resistant mechanisms ,9,10. Rehe world ,13,14,15rom Iran ,17,18,19rom Iran ,21. The patients ,23,24. Bpatients ,26. TherOne of the alternative agents to conventional antibiotics is antimicrobial peptides (AMPs), which have been under focus regarding their potential activities against microorganisms. The AMPs are defined as small molecules (10–50 amino acids) with a positive net charge (+2–+9) that exhibit amphipathic properties. They have critical roles in modulating the innate immune system of the host’s defenses ,28,29.AMPs are generally bactericidal and exhibit broad-spectrum antimicrobial effects against Gram-positive and Gram-negative bacteria via the electrostatic interaction between the negatively charged microbial cell surfaces  and the positively charged peptides ,31. TheyNevertheless, there are some limitations for the clinical applications of AMPs, such as their potential toxicity to human cells, the susceptibility to proteases, and the high cost of industrial peptide (>20 residues in length) synthesis ,37. In oCathelicidins are a major group of cationic antimicrobial peptides and have been detected in the immune system of several vertebrates. The cathelicidin’s structure consists of two different regions: a cathelin-like domain at the N-terminus that displayed a high similarity, at the intra-species and a heterologous domain at the C-terminus that represented the antimicrobial activities . Bungarus fasciatus snake (Cath-BF), which exhibited potential antimicrobial activity against the MDR pathogenic bacteria, with minimal hemolytic and cytotoxic effects on human cells [Recently, a new type of cathelicidin was determined in the venom glands of the an cells ,42. It ian cells ,44. As ran cells . The possible application of developed AMPs as anti-biofilm agents on biomaterial surfaces is useful in the hospital setting. They can exert for combating MDR and/or biofilm forming bacterial infections in the healthcare system . E. coli utilization system. The developed expression systems in E. coli have provided an opportunity to produce large quantities of various AMPs [E. coli host, instability of AMPs against bacterial proteases, the correct fold of expressed products, are challenges to achieve a biologically active form of AMPs. For this reason, several fusion partners have been introduced to facilitate the expression and purification of AMPs [In the present study, Cath-A was used for anti-biofilm tests. In order to establish cost-effective production with the potential activity of the peptide, the recombinant Cath-A (rec-Cath-A) gene sequence was expressed by the ous AMPs ,48,49,50 of AMPs ,39. 7 promoter and TrxA as a fusion partner was applied in order to reduce the toxicity of the AMPs to the host cell and to express the foreign protein in a soluble form [A. baumannii and P. aeruginosa.According to the nature of the expressed peptide, the plasmid of pET-32a with a Tble form . This prd-1-thiogalactopyranoside (IPTG) and chemical reagents were purchased from Sigma Aldrich Co. . The antibiotic disks prepared from MAST House . The colistin disk was prepared from Rosco . The Luria–Bertani (LB) broth and Mueller–Hinton (MH) broth , and the Tryptic Soy Broth (TSB), LB and MH agar  were prepared. The polymerase chain reaction (PCR) reagents and plasmid extraction kit were purchased from Bioneer . All of the chemicals that were used were of the analytical grade. The expression vector pET-32a (+) was purchased from Novagen . E. coli strain DH5α was used for sub-cloning and plasmid amplification. The E. coli Bl21 (DE3) was a host for the expression transformed plasmids. The clinical bacterial isolates were prepared by the Milad Hospital, Tehran, Iran. The ATCC standard controls were kindly donated by the referral laboratory of Iran’s Ministry of Health and Medical Education. The HisTrap FF 5 mL column  was used as an affinity chromatography for the purification of the histidine-tagged recombinant protein.The peptide with a purity ≥90% was synthesized by GL Biochem Ltd. . The antibiotic powders , enterokinase, isopropyl β-P. aeruginosa and A. baumannii isolates were collected for the current study during three months. All of the bacteria were isolated from the clinical instruments  of the patients who were submitted to the intensive care unit (ICU) of the Milad Hospital, Tehran, Iran. The bacterial strains were confirmed by standard microbiological tests. The antibiotic susceptibility tests against the conventional antibiotics were performed by the disk diffusion method based on the Clinical and Laboratory Standards Institute (CLSI) [P. aeruginosa isolates were tested against colistin (10 μg). The minimum inhibitory concentration (MIC) of the isolates was also tested using the broth microdilution method against ampicillin, tetracycline, levofloxacin, ceftazidime, cefepime, piperacillin, and imipenem based on the CLSI recommendation [E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as controls for the antibiotic tests.The MDR e (CLSI) . The strendation . The E. 5 colony-forming unit (CFU)/mL inoculum. The plates were incubated for 24 h at 37 °C. The MH broth with untreated bacterial suspension was used as the positive control. The negative controls were in un-inoculated media with normal saline. The MIC of the peptide was defined as the minimum concentration which inhibited the bacterial growth visually. All of the experiments were performed in triplicates.The antimicrobial activity of the synthetic peptide was tested against the clinical and standard isolates by the modified broth microdilution method as described previously . BrieflyP. aeruginosa no. 1 and A. boumannii no. 1  1× to remove the non-attached cells. The established biofilms were treated with 10 μL of 10× concentrations of antimicrobial agents (Cath-A and levofloxacin) to reach a final concentration of 4–256 μg/mL and 90 μL of fresh MH broth. The bacterial suspension without any treatment agents was performed as the positive control. The plates were incubated for 24 h at 37 °C. Following the incubation time, the plates were washed with PBS so as to remove the planktonic cells and left to dry at room temperature. For the fixation of the biofilms, 100 µL of 100% methanol was added for 10 min, then, by removing the methanol, the wells were stained with 100 μL of 0.1% crystal violet (cv) for 15 min. The TCPs were washed with sterile distilled water and were left to dry. Finally, 100 μL of glacial acetic acid (30% v/v) was added to dissolve the cv. The absorbance of the stained biofilm was measured at an optical density (OD) of 595 nm by a microplate reader . The experiments were conducted in triplicates.Two clinical strains E. coli expression system, the codon optimization was done and the gene was inserted into pET-32a as an expression vector. The recombinant plasmid (pET-32(a)—rec Cath-A) and non-recombinant ) were transformed into competent E. coli DH5α cells. The positive transformed cells were confirmed by the colony PCR. The PCR tests were performed using T7 universal primers. The PCR products were purified with a PCR product purification kit and were sequenced to exhibit the fidelity of the transformed plasmids by an ABI 3130 Genetic Analyzer . The plasmids of the positive competent cells were extracted using a Bioneer plasmid mini extraction kit and were transformed into competent E. coli Bl21 (DE3) cells.The entire gene sequence, including the rec- Cath-A with the additional enterokinase cleavage site was designed, as seen in To optimize the highest protein expression, different media cultures, IPTG concentration, incubating times, and temperatures after the IPTG induction, were tested. E. coli BL21 (DE3) with expression vector was inoculated in 50 mL of the 2XYT medium containing 100 mg/L of ampicillin and were cultured in a shaking incubator at 37 °C for 12 h. Then, 10 mL of the culture was transferred to 500 mL of a fresh Terrific broth (TB) medium containing 100 mg/L ampicillin and 1% glucose. The bacteria were cultured at 37 °C with 200 rpm shaking until the optical density (OD 600) reached 0.6. The protein expression was induced by IPTG to the final concentration of 1 mM. The transformed cells were cultured for a further 6 h of cultivation at 25 °C. Subsequently, the cells were harvested by centrifugation at 5000 rpm, at a temperature of 4 °C for 10 min. The bacterial pellets were re-suspended in the binding buffer  and were lysed on ice by sonication for 10 cycles (20 s working and 40 s rest). The supernatant of the lysate was collected by centrifugation at 14,000 rpm for 20 min at 4 °C. The extracted proteins were detected by a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and were stained with coomassie brilliant blue R-250.A fresh colony of transformed 2PO4, 500 mM NaCl, 500 mM imidazole; pH 7.5). The peak fractions were collected and concentrated overnight at 4 °C by polyethylene glycol (PEG) with a 20,000 molecular weight. The concentrated proteins were analyzed on SDS-PAGE.The proteins were applied to His Trap FF columns in order to detect the His-tag proteins. The column was washed with the binding buffer several times. The extracted proteins were loaded on a column and were eluted by a gradient of the imidazole concentration (0–100%) from an elution buffer (20 mM NaH2) overnight at 4 °C.The fraction, which contained the recombinant peptide (rec-Cath-A) were dialyzed against a 500-mL volume of the enterokinase buffer  with 30 kDa as well as a 3 kDa molecular weight cut-off in order to remove the enterokinase and non-specific proteins. The peptide solution was dialyzed (14 kDa cut-off) in 5 mL of PBS the 1X buffer in order to achieve a released Cath-A peptide and was analyzed by SDS-PAGE.The rec- Cath-A fusion protein was incubated with enterokinase at a ratio of 5 U:1 µg of the fusion protein in 10 mM of Tris HCI, pH 8.0, and 10 mM CaClThe standard Bradford protein assay was displayed for the quantitative determination of the purified protein concentration .The antimicrobial activity of the purified peptide was determined using the agar disk diffusion method, based on the standard assay which was recommended by the CLSI against standards and clinical MDR bacteria . Briefly, the 0.5 Mcfarland of the fresh colonies of bacteria were sub-cultured on MH agar and blank disks with different concentrations of synthetic peptide were placed on the plate. Approximately 20 μL of the dialyzed peptide was inoculated on the blank disk. The plates were incubated at 37 °C overnight. The inhibition zones were measured and compared to the synthetic peptide. All of the experiments were performed in triplicates. p values < 0.05 and 0.01 were indicated as significant. The experiments were performed in triplicates. Data were analyzed by one-way analysis of variance (ANOVA), and standard deviations of the mean (mean ± SD) were presented for each test. P. aeruginosa isolates that were susceptible to colistin in the disk diffusion method . The average inhibitory concentration effect of the peptide (134 μg/mL) was greater than levofloxacin (65 µg/mL) and imipenem (82 µg/mL) for clinical P. aeruginosa.Clinical isolates exhibited resistance to all antibiotic disks except n method . The MICP. aeruginosa 1 and A. baumanii 1 without mucoidal surfaces and a level of resistance to levofloxacin were collected for the biofilm formation in polystyrene plates. After the biofilm formation and antimicrobial treatment, the plates were stained by 0.1% cv and OD 595 nm was read by a micro-plate reader. The amounts of OD revealed the adhered bacterial biomass. According to these findings, among the untreated bacteria, the growth of P. aeruginosa was much higher than A. baumannii.The effects of Cath-A and levofloxacin against biofilm structures formed by the MDR isolates in the TCP assay are displayed in p < 0.05). Cath-A could remove a considerable amount of the attached biomass of P. aeruginosa in comparison to levofloxacin at concentrations ≥128 µg/mL (p < 0.01). With respect to this analysis, Cath-A could reduce the A. baumannii biofilm at lower concentrations. The antibiofilm effects of the peptide to decrease the A. baumannii biomass were considerable at ≥8 µg/mL concentrations. The Cath-A peptide could eradicate almost all of the A. baumannii attached cells in the biofilm at concentrations ≥64 µg/mL, in contrast to levofloxacin.The activities of Cath-A and levofloxacin to remove the planktonic cells were significant compared to the control cells (E. coli BL21 (DE3) for the expression of the fusion protein by the T7 promoter. Time and medium compositions were major factors which were evaluated to achieve large scales of production yields. The soluble products were expressed in the TE medium after 6 h of 1 mM IPTG induction at 25 °C. Increasing the time factor to more than 6 h after IPTG induction, led to the decrease of the peptide production. The expression of the constructed vector including the recombinant peptide (~22.8 kDa) in various expression conditions was analyzed by SDS-PAGE (The construct of the recombinant plasmid (pET 32a +rec-Cath-A) with (approximately) a 22.8 kDa molecular weight, including the non-toxic fusion protein (Trx), His-tag sequence for affinity chromatography, enterokinase cleavage site the for peptide release, and the Cath-A sequence  was confSDS-PAGE . Using the HisTrap FF affinity column, soluble fusions including Trx–6His–enterokinase–Cath-A were purified in an elution buffer containing 0.5 M imidazole. To detect exactly the fraction that contained rec–Cath-A, a gradient of the imidazole concentration in the elution buffer was used. According to the results, two fractions were eluted at 17% (85 mM) and 47% (235 mM) of the imidazole concentration. The fractions were concentrated using PEG and were analyzed by SDS-PAGE . The desE. coli ATCC 25922, P. aeruginosa ATCCC 27863, and clinical isolates of P. aeruginosa and A. baumanii . Additionally, the cell culture and hemolysis studies proved that the peptide was safe in its effective concentrations [An imminent need for new antibiotics dictates the necessity for studies aimed at designing clinically useful AMPs. The present study was carried out on such a basis and it provides a 34 amino-acid long AMP sequence named Cath-A. In previous reports, this peptide was proven to be effective in ex vivo studies using different pathogen microorganisms containing standard and wild-type strains, especially the methicillin-resistant trations . Bacterial cells form biofilm matrixes under several environmental conditions including nutritional signs and starvation, attachment to the host tissues or non-living surfaces, exposure to sublethal concentrations of antibiotics, and environmental stresses. The biofilm structures are resistance to the stress, especially the conventional antimicrobial agents and the host defense mechanisms. It is estimated that microbes growing in the biofilm structures are more resistant to antibiotics compared to the planktonic cells. So the treatment of biofilm-related infections is a significant problem in healthcare systems. To clear the biofilm forming bacteria, the various antibiotics classes  are generally used. Hence, new developed antibacterial agents with alternative strategies are required for the treatment and eradication of established biofilms in clinical settings ,58,59. AA. baumannii and P. aeruginosa biofilm isolates. Among the opportunistic bacteria, MDR P. aeruginosa and A. baumannii are considered medically important pathogens. These pathogens are commonly associated with nosocomial and hospital-acquired infections, especially in the ICU and burn sites. [A. baumanii and P. aeruginosa [A. baumanii isolates. In the case of the P. aeruginosa strains, Cath-A had anti-biofilm effects comparable to commercially available antibiotics. However, some isolates among P. aeroginusa exhibited a resistance to Cath-A (MIC ≥ 265 µg/mL) which may be related to the bacterial resistance mechanisms such as proteolytic degradation, the modification of cell wall components, or the presence of alginate in the biofilm structure [Therefore, in this report, the antimicrobial potential of Cath-A was examined against the n sites. ,60,61. Tn sites. . Other rruginosa ,63,64. Mruginosa ,66,67. Ttructure . AdditioE. coli expression system was applied so as to express recombinant Cath-A because of its cost-effectiveness and the over-construction of the desired product. The suitable expression vector (pET 32a) was used as it contains the Trx fusion protein in order to eliminate the toxicity of the peptide and to express the soluble peptide in a correct folding. The existence of the His-tag was an important factor in the purification process using the affinity chromatography from the HisTrap FF column. These properties made the purification easier and increased the product yield. The inserted extra enterokinase sequence at the N-terminus of rec-Cath-A facilitated the cleavage process and ended up being the desired sequence. In the present study, the time of expression after the IPTG induction played a critical role in producing rec- Cath-A. This is in accordance with the reports of other researchers [E. coli [Another important issue in this report was the method that was used for the production of Cath-A. The earchers ,70. In c[E. coli . This ma[E. coli ,73.E. coli as an expression host. The potential antimicrobial effect of Cath-A against MDR bacteria was demonstrated. Our findings suggest that the Cath-A peptide could be a promising topical candidate to control the MDR biofilm-forming bacteria in the healthcare settings.In this paper, we have described an expression approach to produce soluble, active, and partially purified recombinant Cath-A in


Purpose: This study tried to explore whether members of miR-92a family contribute to early diagnosis and prognosis for human cancers and how they work.Methods: Integrated meta-analysis retrieved from public repositories was employed to assess the clinical roles of the miR-92a family for cancer diagnosis and prognosis. Expression level of miR-92a was detected by the TCGA database and was confirmed by non-small-cell lung cancer (NSCLC) tissues. Targets of miR-92a were predicted using starbase, and validated by dual luciferase assay. Correlation between miR-92a and the target gene was assessed by linkedOmics while expression of the target gene and its role in cancer prognosis were analyzed with UALCAN and Gepia.Results: We recognized the miR-92a family could serve as a potential diagnostic biomarker with a pooled sensitivity of 0.85 [0.81–0.88] and specificity of 0.86 [0.83–0.90]. The overall hazard ratio (HR) was 2.26 [95% CI: 1.70–3.00] for high expression groups compared to low expression groups. Expression of miR-92a was identified to be upregulated in NSCLC, especially in lung squamous cell carcinoma (LUSC). Results from starbase and dual luciferase assay indicated the regulator of G-protein signaling 3 (RGS3) was a direct target of miR-92a. Statistical negative correlation was found for the expression of miR-92a and RGS3. In addition, expression of RGS3 was downregulated in NSCLC and patients with the high expression had a poor prognosis (HR = 1.3) for LUSC patients. However, results were to the contrary for lung adenocarcinoma (HR = 0.7).Conclusion: This study revealed that miR-92a family could be ideal biomarkers for cancer diagnosis and prognosis, which might function through targeting RGS3. MicroRNAs (miRNAs) are small, conserved, single-stranded, non-coding RNAs, which adjust gene expression in the post-transcriptional level . The search strategy was (hsa-miR-92a or miR-92a or microRNA-92a or miR92a or hsa-miR-25 or miR-25 or microRNA-25 or miR25 or hsa-miR-363 or miR-363 or microRNA-363 or miR363) and (tumor or carcinoma or neoplasm or cancer), which were updated until March 10, 2018. The reference list was also retrieved. Criteria were drafted for literature screening. Demographic information and data for meta-analysis including sensitivity (SEN), specificity (SPE), true positive (TP), false positive (FP), false negative (FN), true negative (TN), HR with 95% CI was collected from included literature by two authors (MJ and XY-L) independently. The quality of publications with diagnostic data was evaluated based on the Quality Assessment of Diagnostic Accuracy Studies 2 guidelines (QUADAS-2), while guidelines from the Newcastle-Ottawa Scale (NOS) were followed for quality assessment of publications with prognostic data Stang, .http://ualcan.path.uab.edu)    tissues, 503 LUSC tissues and 111 normal tissue samples) downloaded from TCGA was used to evaluate the expression level of miR-92a and the expression of target gene by UALCAN  tissues with adjacent normal tissues were obtained from 52 patients with NSCLC between July 2010 and December 2014. All tissues were quick-frozen in liquid nitrogen and stored at −80°C. All patients provided the informed consent. This research was approved by the Institutional Review Board of China Medical University.2.The human NSCLC cell line A549 was purchased from GeneChem  and was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum (FBS) at 37°C with 5% COFor overexpression of miR-92a, the cells were transiently transfected with 100 nM miR-92a mimics and negative controls synthesized by JTS  using jetPRIME . For overexpression of RGS3, 1 μg RGS3 vector and the negative control  were transfected using jetPRIME as described above.−ΔΔCt method.Total RNA was exacted from tissue samples using RNAiso Plus  following the manufacture's procedure. To detect the RNA expression levels of miR-92a, TaqMan MicroRNA Reverse Transcription Kits  were used for cDNA synthesis and TaqMan MicroRNA Assays  were used for quantitative PCR. U6 was used as an internal control. The PCR was repeated three times for every sample. The relative expression was calculated by the 2Starbase v2.0 . The potential binding sites in 3′-UTR of RGS3 were inserted into the Dual-Luciferase vector. A vector containing the mutant 3′-UTR fragment of RGS3 was constructed as a negative control. The luciferase reporters were co-transfected with miR-92a mimics or negative control by jetPRIME . At 48 h after transfection, the luciferase activities were detected with the Dual-Luciferase Reporter Assay System (Promega) in Synergy H1 system (Bio Tek).I2 value and Q-test . RevMan 5.3 software (version 1.4) was employed to assess the quality of enrolled literature for diagnostic meta-analysis. While all other statistical analyses were conducted using SPSS 20.0 . The t-test was performed to compare the means of two groups. Analysis of variance was conducted to compare the results among three or more groups. Dunnett-t-test was employed to compare the results between different experiment groups with a same control group. Spearman correlation analysis was used to assess the correlation between expression level of miR-92a and the target gene. A value of P < 0.05 was defined as indicating statistical significance with two-tailed.Overall diagnostic estimates including SPE, SEN, negative likelihood ratio (NLR), positive likelihood ratio (PLR) and the diagnostic odd ratio (DOR) were assessed by the random effect regression model. Summary receiver operating characteristic (SROC) and value of area under the SROC curve (AUC) were constructed and evaluated. A detailed diagnostic analysis was conducted including meta-regression analyses and subgroup analyses  to identify and decrease the heterogeneity. Moreover, the pooled HR with its 95% CI was evaluated for prognostic meta-analysis. Simultaneously, heterogeneity among these publications was analyzed by P-value equal to 0.92 for Deek's funnel plot asymmetry test indicated that there was no publication bias among these researches , indicating that low-RGS3-expression groups had a higher risk of death probability than the high-expression groups. However, the opposite result was obtained for LUSC as shown in To detect the expression level of RGS3 in NSCLC, we downloaded the mRNA expression dataset of RGS3 in NSCLC from TCGA. Results indicated that the expression of miR-92a was low in NSCLC compared with normal tissues  of poor outcomes compared to groups with low expression. The group with an upregulated expression of miR-25 and showed an increased risk with respect to disease progression or mortality with the corrected HR of 2.22 [95% CI: 1.67–2.97]. Nevertheless, the overall HR was 2.11 [95% CI: 0.96–4.65] with a Z value of 1.85 and a Since miRNA family members have the same seed area, all family members may have the same target gene. Therefore, we chose the miR-92a as the representative of this family to conduct the follow-up mechanism research. As the result of our research, the expression level of miR-92a was downregulated in LUSC in the TCGA dataset, which was confirmed in NSCLC tissue analysis. We further identified RGS3 as a target gene of miR-92a in lung cancer. The luciferase assay demonstrated that miR-92a could directly bind to the 3′-UTR to decrease the expression of RGS3, one of the “cancer signature” genes owing to its vital role in tumor development. RGS is a GTPase-activating protein, which could directly interact with the alpha subunit of G protein, and then negatively regulate G protein signal conduction by catalyzing the hydrolysis of GTP (Scheschonka et al., P < 0.001] when compared to lower expression groups (Qu et al., As far as we know, this research is the first one to summarize the clinical value of the miR-92a family on cancer diagnosis and prognosis though there have been several meta-analyses data published that focused on the correlation between a single miRNA and its clinical application. In one study dedicated to the role of miR-92a in colorectal cancer diagnosis, the authors drew a conclusion from only six publications, suggesting miR-92a might be a biomarker for colorectal cancer with moderate detection ability, with SEN and SPE of 0.76 [0.72–0.79] and 0.64 [0.59–0.69], respectively (Yang et al., Despite the above efforts and advantages of this study, some limitations in the present study still need to be addressed. The first and most important one was heterogeneity among the included articles, which could not be neglected for any meta-analysis, and which might potentially influence the final results. Therefore, we employed the meta-regression and subgroup analysis with a random effect model to reduce or avoid the influence of heterogeneity. Secondly, due to insufficient data on cancer types and studies, subgroup analyses of prognostic meta-analyses could not be conducted. Moreover, some studies might have been omitted in the literature selection process. All these above considerations may lead to negative conclusions; therefore, further larger, long-term follow-up studies are needed to obtain robust and possibly definitive evidence.In summary, this study demonstrated that serum miR-25 might play an important role in a clinical setting for cancer early diagnosis and prognosis prediction, especially for lung cancer in an Asian population; while tissue miR-92a may be a suitable biomarker for cancer detection, and this family might function through targeting RGS3.All datasets generated for this study are included in the manuscript/The studies involving human participants were reviewed and approved by Institutional Review Board of China Medical University. The patients/participants provided their written informed consent to participate in this study.BZ and MJ planned the research. MJ and XiL retrieved literatures. MJ finished the experiments. XuL and XQ conducted statistical analysis. BZ, XuL, and MJ wrote and edited this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fluorescence/temperature-sensitive hydrogels, thanks to their properties in fluorescence and temperature sensitivity, have shown a promising outlook in the fields of drug delivery, cell imaging, etc., and thus become the focus of present research. This paper reports the preparation of green-fluorescence/temperature-sensitive hydrogels through one-step radical polymerization with green fluorescence-emissioned carbon dots as fluorescence probes and N-isopropylacrylamide as a monomer. UV-vis spectra, fluorescence spectra, and fluorescence microscope imaging have been used to characterize the prepared hydrogel, and to study their optical and temperature-sensitive properties. It was discovered that the emission of prepared hydrogel is excitation wavelength-dependent, pH responding, and excellent temperature-sensitive, as well as having good biocompatibility. The prepared hydrogel can also be applied as fluorescence ink in the fields of anti-counterfeit identification and appraisal. Thanks to its excellent biocompatibility ,2, opticCarbon dots (CDs) have attracted widespread attention from the researchers thanks to their heavy-metal free, raw material richness, simple synthesis process, excellent optical stability and biocompatibility, and low toxicity ,19,20,21This paper reports the one-step preparation of CDs/PNIPAM fluorescence/temperature-sensitive hydrogel with G-CDs, acrylic acid (AAc) and NIPAM as raw materials, N,N’-methylenebisacylamide (BIS) as a crosslinking agent, and potassium persulphate (KPS) as the initiator through polymerization reaction . The typical properties of prepared CDs/PNIPAM, such as optical properties, temperature sensitivity, and dependence on pH environment, have been studied. The prepared hydrogel has been applied in fluorescence imaging, ink, and anti-counterfeiting identification. This research would provide new material for a wide range of fields like temperature-sensitive sensors, drug carrier system, fluorescent labeling, fluorescent coding, and fluorescent ink.1800) were obtained from Aladdin Chemistry Co., Ltd. ; acrylic acid (AAc), potassium persulphate (KPS), L-ascorbic acid (AA), citric acid monohydrate (CA), and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co., Ltd. ; cyclohexane was obtained from Tianjin Bodi Chemical Co., Ltd. ; and acetone was purchased from Tianjin Fuyu Fine Chemical Co., Ltd. . Tris–HCl buffer solutions with different pH values were prepared by dropwise and added into a concentrated hydrochloric acid solution (0.1 mol·L−1) or tris solution (20 mmol·L−1) to required pH values. L02 cell was purchased from Tongpai Biological Technology Co., Ltd. . All chemicals used were of analytical grade or of the highest purity available. Deionized water was prepared from a Milli-Q-RO4 water purification system .N-isopropylacrylamide (NIPAM), N,N’-methylenebisacrylamide (BIS), and ethylene imine polymer  to determine the bandgap absorption of CDs/PNIPAM. Fluorescence spectra were recorded on a LS55 spectrofluorometer . Experiments on the fluorescence emission as a function of temperature were conducted, ranging from 25–43 °C. The pH was monitored by a PHSJ-3F pH meter . Zeta-potential measurement was carried out on a Zetasizer nanoseries ZEN3690 . Images of fluorescent ink were lit on a ZF-1 three-use UV analyzer . The TEM images were obtained at 310 K magnification with a FEI Tecnai G2 20 s-twin transmission electron microscope . Fluorescence lifetime was measured with an FLS920 fluorescence spectrometer . Microscope images were acquired with the XD30-RFL microscope .1800, and 0.8 g of CA were dissolved in 10 mL of distilled water, and the mixture was stirred at room temperature for 5 min. The solution was then transferred to an autoclave and further reacted at 150 °C for 1 h.G-CDs used in experiments were synthesized by our laboratory . A measuBoth NIPAM and BIS samples must be recrystallized and purified before use. A measure of 0.124 g of NIPAM, 0.0062 g of BIS, and 21 μL of AAc were dissolved in 13.04 mL of deionized water within the flask equipped with a mechanical stirrer and purged with nitrogen for 30 min at 70 °C. The polymerization was initiated by the addition of 0.02 g of KPS dissolved in 0.5 mL of water. The solution was heated to 70 °C with nitrogen for 30 min. After the flask was sealed, the reaction was continued for another 4 h before being cooled down naturally. After cooling, the microgels were purified by ultrafiltration at 8000 rpm for 10 min, and dispersed in deionized water. The PNIPAM hydrogels were obtained.A measure of 0.124 g of NIPAM, 0.0062 g of BIS, 21 μL of AAc, and 1 mL of G-CDs were dissolved in 13.04 mL of deionized water within the flask equipped with a mechanical stirrer and purged with nitrogen for 30 min at 70 °C. The polymerization was initiated by the addition of 0.02 g of KPS dissolved in 0.5 mL of water. The solution was heated to 70 °C with nitrogen for 30 min. After the flask was sealed, the reaction was continued for another 4 h before being cooled down naturally. After cooling, the microgels were purified by ultrafiltration at 8000 rpm for 10 min, and dispersed in deionized water. The CDs/PNIPAM hydrogels were obtained.4 cells per well for 24 h. The sample of CDs/PNIPAM was dissolved in deionized water, and DMEM medium was added to prepare different concentrations. Different concentrations of diluted samples were added to each well (DMSO concentration was less than 1‰). The cells were incubated for a further 24 h. After the supernatant was discarded, 100 μL of MTT solution was added to each well, and the mixture was placed in an incubator for a further 30 min. The supernatant was discarded and 150 μL of DMSO was added to each well. They were shaken in the dark for 10 min. The microplate reader detected the optical density (OD) at 562 nm.To analyze the in vitro cytotoxicity of CDs/PNIPAM, L02 cells were seeded in 96-well plates with a density of about 1 × 10The fluorescence probe used in this research is the G-CDs with PEI as the carbon source, and their UV-Vis and fluorescence spectra are shown in As shown in The fluorescence microscopy imaging of prepared CDs/PNIPAM hydrogel under different excitation wavelength was investigated. As shown in 1 and τ2 are decay time constant, and A1 and A2 are the ratios of time resolved decay lifetime that τ1 and τ2 take. The average fluorescence lifetime of CDs/PNIPAM (τavg) hydrogel was calculated according to the equation:The fluorescence lifetime was then tested according to the double exponential function of nonlinear least squares analysis to fit the fluorescence decaying curve of prepared CDs/PNIPAM hydrogel:1) and a long lifetime (τ2) for CDs/PNIPAM hydrogel. The double exponential behavior of fluorescence lifetime shows the existence of two different emission positions (fast decay component), corresponding to the recombination of surface state and molecular state. Compared with molecular state, the emissions originating from surface state usually exhibit shorter recombination lifetime. Therefore, it can be inferred that τ1 originates from the surface state of CDs of the surface, while τ2 is from the molecular state, and the average lifetime is calculated to be 8.21 ns.According to the fitting result in The TEM image of CDs/PNIPAM hydrogel is shown in 4+ on the surface of prepared G-CDs, making it positively-charged in this environment. The zeta potential of PNIPAM hydrogel is −14.4 mV because of the carboxyl group in the raw materials of hydrogel . The zeta potential gets an obvious decrease (−3.36 mV) in CDs/PNIPAM hydrogel because the positive-charged G-CDs were embedded into the hydrogel, leading to the decrease of absolute potential. The surface potential of nano-particles can be measured and analyzed by zeta potential. The zeta potentials of G-CDs, PNIPAM hydrogel, and CDs/PNIPAM were measured separately. As can be seen from −1 originates from the stretching vibration of N–H, and the peak at 2980 cm−1 originates from the stretching vibration of C–H; the peak at 1720 cm−1 is from the —CONH-group, and the peaks at 1640 and 1560 cm−1 are the stretching vibration of C=O and the in-plane bending vibration of N–H. The characteristic peaks of PNIPAM hydrogel are basically in accordance with those of CDs/PNIPAM hydrogel. The absorption peaks at 1420 and 1220 cm−1 originate from C–H and C–O/C–O–C groups from G-CDs. This shows that G-CDs do not only exist on the surface of prepared CDs/PNIPAM hydrogel, but also are embedded inside the hydrogel. The CDs/PNIPAM hydrogel is successfully prepared.FTIR was used to characterize the functional groups on the surface of CDs/PNIPAM hydrogel. As shown in ex = 480 nm). As shown in Since temperature sets an obvious impact upon the fluorescence properties of prepared CDs/PNIPAM hydrogel, the temperature-sensitive performance of the hydrogel was investigated. The temperature of CDs/PNIPAM hydrogels was controlled in the range of 25–43 °C through water-bathing to study the fluorescence intensity change with temperature (λTo investigate the reversibility and repeatability of the temperature-sensitive fluorescence of CDs/PNIPAM hydrogel, the hydrogel was heated to 43 °C and then cooled down to 25 °C six times, and the fluorescence spectra were measured. As shown in Before studying the fluorescence intensity of CDs/PNIPAM hydrogel in different pH environments, the stability of G-CDs was first examined. As shown in As shown in Good biocompatibility is a prerequisite for the application of CDs/PNIPAM hydrogels to drug delivery. The biocompatibility of CDs/PNIPAM hydrogels was examined by an MTT assay. As shown in The CDs/PNIPAM prepared herein could be applied to fluorescent inks. The hydrogel ink was written on filter paper and photographed under a 365 nm UV lamp. As shown in This paper reports the synthesis of CDs/PNIPAM hydrogel with excellent fluorescence property and temperature-sensitivity. The hydrogel was prepared through a one-step route under low temperature, with long wavelength-emissioned G-CDs as fluorescence probe and NIPAM as monomers. The optical characterization experiments show that the prepared hydrogel exhibits classic excitation wavelength-dependent photoluminescence properties. The experiments also show that the hydrogel exhibits sensitive response to temperature with excellent reversibility and repeatability, and it also exhibits certain pH-response property and excellent biocompatibility. The prepared CDs/PNIPAM hydrogel can be used as fluorescent ink, and it emits clear and bright fluorescence under UV radiation. This composite hydrogel provides new fluorescence/temperature-sensitive material for the fields of portable temperature-sensitive sensors, drug carrier, cell imaging, fluorescent ink and anti-counterfeit identification.


BC maintained the graft space and released BMP-2 in a sustained manner, promoting optimal bone formation. The BC+BMP-2 combination enhanced bone regeneration and shows promise as a useful means of clinical pre-dental implant bone augmentation in the maxillary sinus.If the alveolar bone height of patients requiring dental implants in the maxillary molar region is inadequate, it is difficult to achieve satisfactory outcomes using existing bone graft materials. We previously reported the possible utility of bacterial cellulose (BC) as a new dental treatment material. BC has a high absorptive capacity, good mechanical strength, and good volume retention. BC loaded with bone morphogenetic protein-2 (BMP-2) might allow effective alveolar bone augmentation. We created critical frontal bone defect models in 12 male Japanese white rabbits and divided them into four groups: sham; BC (BC grafting only); BMP-2 (treated with BMP-2 solution only); and BC+BMP-2 (grafted with BC loaded with BMP-2). Newly formed bone volume was calculated via hematoxylin-eosin staining evaluation. The proliferating cell nuclear antigen and osteocalcin levels were determined by the immunohistochemical staining analysis. All measured indices of the BC+BMP-2 group were significantly superior to those of the other groups (all  Although dental implantation is now a routine procedure, placing implants in the posterior maxillary region remains a challenge because the height of the alveolar process is often insufficient due to the location of the maxillary sinus. To resolve this anatomical constraint, Boyne and James ,2,3 deveIn the future, bone augmentation methods will need to incorporate alternative bone graft substitutes and enhance bone regeneration. Although various bone graft substitute methods have been explored, the outcomes have often been unsatisfactory because it is difficult to retain the bone’s three-dimensional structure ,4,5,6,7.Bacterial cellulose (BC) is produced by certain strains of bacteria and differs substantially from plant cellulose. BC is a thin filamentous fiber approximately 1 μm in diameter that intertwines with itself and forms a crystalline, high-elasticity, and superabsorbent net-like structure. BC is sturdy, retains its shape, has a high-water content, and has many industrial applications in foodstuff preparation ,16,17,18Bone morphogenetic proteins (BMPs) are promising osteoinductive substances facilitating ectopic bone formation. When BMPs are loaded into a carrier and placed under muscle or skin, bone growth is induced, even in an area where there was no previous bone growth. This is achieved through enhanced differentiation of mesenchymal cells to osteoblasts and chondroblasts ,21,22. HBMP-2 is the best-studied bone-forming protein. Currently, BMP-2 is the only bone morphogenetic protein approved as a bone graft substitute . In oralAcetobacter hansenii  were purchased from the American Type Culture Collection . BC sheets  were obtained by culturing Acetobacter hansenii in 10-cm laboratory dishes containing CSL-Frc medium at 28 °C and a pressure of 101.3 kPa (20 vol% oxygen) for approximately three days until a 1-mm thick gel formed. The wet gel BC sheets were autoclaved three times for 20 min at 105 °C in deionized water, freeze-dried , and compressed to a thickness of 0.5 mm using a small press  [Two strains of , Japan) . The com, Japan) .A stock human-derived recombinant BMP-2 (rhBMP-2) solution (1.0 µg/µL)  was diluted to 0.5 μg/μL with sterilized distilled water  .Ten microliters of rhBMP-2 solution (0.5 μg/μL) was carefully loaded onto the surface of each side of the BC sheet using a micropipette. Therefore, all BC sheets were loaded with 5 μg of rhBMP-2. As in the study by Silvestre et al. , SEM imaTwelve male Japanese white rabbits  were purchased from BioTek . The animals were acclimated for one week at standard conditions  in a room that was ventilated at a rate of 13 times/h. They were fed ORC-4  and had free access to filtered water.The rabbits were anesthetized with 80 mg/kg ketamine HCl given intravenously  and 10 mg/kg xylazine was given intramuscularly in the rump . The fur over the surgical field was shaved, the skin was sterilized with 70% ethanol, and 0.3 mL of 2.0% lidocaine with 1/80,000 epinephrine  was infiltrated. Using a no. 11 surgical knife, a head arc incision extending under of periosteum was made and a skin periosteal flap was created A. The peAfter grafting, no surgical complications occurred and no visual signs of inflammation or infection were observed. The four rabbits from each group were sacrificed at four and eight weeks by injecting 40 mg/kg pentobarbital sodium into a large auricular vein . A thoracoabdominal skin incision was made and the sternum was removed to expose the heart. The left ventricle was cannulated, and the right atrium was cut open. Reflux blood removal was performed with saline containing heparin.All animal experiments followed the guidelines for humane care and use of laboratory animals of the Shimane University Faculty of Medicine, and the study protocol was approved by our Institutional Animal Ethics Committee .v/v) acid ethanol  and then rinsed in distilled water. Next, the sections were stained with eosin solution  for 3 min, followed by dehydration in graded alcohol  baths and clearing in xylene . The slides were finally examined under a light microscope . The specimens were fixed in 20% phosphate-buffered formalin solution , decalcified with ethylenediaminetetraacetic acid (EDTA), embedded in paraffin, and cut with a microtome  into 5-μm-thick slices. All slices were stained with hematoxylin solution  for 5 min followed by five dips in 1%  monoclonal antibody  and primary mouse anti-proliferating cell nuclear antigen (anti-PCNA) monoclonal antibody  to examine osteoblast differentiation and cell proliferation. Sections were deparaffinized with xylene substituted for ethanol for hydrophilization, and antigens were activated in an autoclave  (at 80 °C for 8 min) using citrate buffer and distilled water before annealing to room temperature over a 12-h period. The specimens were incubated overnight at 25 °C with mouse anti-OC monoclonal antibody and mouse anti-PCNA monoclonal antibody as primary antibodies. After incubation, the secondary antibody was added at 25 °C and reacted with an EnVision kit  for 1 h. The color was intensified using DAB solution  and counterstained with hematoxylin. The slices were then observed under a light microscope .Tissue images were obtained using a microscope at 20× magnification. A digital image of the entire bone defect was constructed by integrating the images. All images were analyzed with the aid of ImageJ software  and the extent of newly formed bone was estimated as the ratio of the area of newly formed bone to that of the entire bone defect as revealed by H and E staining ,29.After immunohistochemical staining for PCNA and OC, we photographed all slices at 40× magnification. Three visual fields were selected in the (randomly chosen) upper, lower, right, or left quadrants surrounding the grafts in the BC and BC+BMP-2 groups. For the BMP-2 and sham groups, we randomly selected three areas between the bone wall and sinus membrane. We calculated the PCNA-positive cell ratio of target cells to the entire number of cells within the visual field. The target cells were osteoblasts for estimating bone formation and fibroblasts for estimating wound healing at the bone augmentation area; inflammation-related cells were excluded. The OC-stained area ratio was the ratio of the stained area to the total area.t test to compare the newly formed bone values, the PCNA-positive cell ratios, and the OC-stained area ratios among groups. Independent-samples t-tests were used to compare the newly formed bone values, PCNA-positive cell ratios, and OC-stained area ratios among different time points. All statistical analyses were performed using SPSS ver. 24.0 . A p-value < 0.05 indicated statistical significance.We used a one-way analysis of variance and the LSD-To evaluate bone formation after grafting, decalcified sections of frontal critical bone defects were subjected to H&E staining. No inflammatory reaction was observed at any time in any group. Either no, or only a small amount of, regenerated (newly formed) osteoid tissue was observed between the frontal bone wall and the sinus membrane in the sham group at any time: clear gaps were evident between the wall and membrane. The extent of new bone growth in the BMP-2 group was slightly greater than that in the sham group: gaps in the connective tissue and newly formed bone were filled gradually. In the BC group, connective and fibrous tissue filled gaps between the BC and sinus membrane at four weeks, and unmineralized new bone and cartilage-like tissue were evident between the bone wall and the BC. However, at eight weeks, regardless of the side, the newly formed bone obviously surrounded the BC. In the BC+BMP-2 group, newly formed bone and cartilage-like tissue surrounded the BC, and clumps of chondrocytes and osteoblasts invaded the BC+BMP-2 composite at four weeks. At eight weeks, abundant mature calcified new bone bonded strongly to the BC+BMP-2 sheet. Bone lacunae hosting mature osteocytes were evident in the mature calcified new bone. Notably, at eight weeks, the extent of new mature bone growth was substantially greater than that in any other group at any time .p < 0.05). Additionally, the quality of newly formed bone in the BC group was superior to that in the BMP-2 group; BC was more significant in terms of osteogenesis than was BMP-2. However, no significant difference was apparent between the BMP-2 and the sham groups (p > 0.05), indicating that BMP-2 alone did not significantly enhance osteogenesis (p < 0.05)  B.p < 0.05, p > 0.05, p < 0.05, PCNA-positive cells (which stained dark brown) were detected in all sections . Howeverp < 0.05, p < 0.05, Similar trends were apparent at eight weeks. Unlike the sham group, the BC, BMP-2, and BC+BMP-2 groups all exhibited significant improvements compared to four weeks  evidenced larger OC-positive areas than at four weeks  at either four or eight weeks. No histological sign of chronic inflammation was apparent. No capsule formation developed around BC or the BC+BMP-2 composite. No foreign body reaction was detected in tissue around BC. When evaluating the long-term outcomes of an implanted scaffold, it is essential to exclude initiation of a chronic inflammatory response. We found that BC exhibited excellent biocompatibility to eight weeks after placement, these results are also consistent with previous studies .Acetobacter hansenii; during biosynthesis, BC forms a pellicle containing a random microfibrillar network of cellulose chains interspersed with amorphous regions [In the BC-only group, at four weeks, connective tissue and fibrous tissue were present around the BC. However, at eight weeks, the tissue was much more mature and organized, featuring a small amount of newly formed ectopic bone tissue surrounding the BC . This he regions . BC cont regions ,34. The  regions . BC is a regions . A graft regions . Thus, wHumans encode 20 BMPs; after birth, BMPs are associated with the collagenous extracellular matrix, being expressed by both periosteal and mesenchymal cells of the marrow stroma during fracture repair ,39. The PCNA and OC status was used to evaluate bone regeneration and cell viability. PCNA is a nucleoprotein of molecular weight of 36 kDa expressed from late G1 to S phase . PCNA isA larger OC-positive area was associated with higher PCNA-positivity in all experimental groups, especially in the BC+BMP-2 group. PCNA is not an independent prognostic indicator of cell activity; rather , PCNA iNotably, the PCNA and OC expression trends were highly consistent with our histomorphological data. We predicted that new bone formation would commence relatively early. It appears that a sustained-release of BMP-2 is effective for enhancing osteogenic cell differentiation, and thus increasing the formation of new bone. Both the BC and BMP-2 groups had more osteoblast differentiation and proliferation than the sham control, but the two groups were similar in these contexts especially at an early bone healing stage. Thus, both a physical structure and chemical stimulation are essential to promote healing. After BC was loaded with BMP-2, healing and bone regeneration were remarkably accelerated. BMP-2 is known to stimulate bone regeneration. However, further study is needed to investigate whether BMP-2 loaded into BC stimulates tissue healing in a manner similar to BMP-2 alone.+-free electrolyte fluid, and electrolyte fluid. Cumulative release of trypan blue revealed that, within the initial 3 min, the drug was released rapidly and achieved a comparatively high level at 6 h; subsequently, the release rate tended to be stable and lasted more than seven days. No significant difference in modulus for BC was found between six hours and seven days [In our previous study, we tested the drug release of BC by loading with saline, Kven days . The timven days . Freire ven days ,48. Howeven days . NeverthBy varying the bacterial culture time, it is possible to produce BC of various fibril densities, which are associated with different properties appropriate in particular circumstances . In prevBC is a versatile biomaterial exhibiting excellent biocompatibility, and can integrate with host tissue without provoking chronic inflammation. These properties were well-demonstrated in our present study. However, this study spanned only eight weeks, so more research is needed to investigate how bone derived by grafting changes over time. It will also be necessary to examine concentration-dependent bone formation by BMP-2 in order to find the most effective dose ,26. ManiThe histological and immunohistochemical evaluations of the present study confirmed the excellent biocompatibility of BC in a rabbit frontal sinus model. Combined with BMP-2, BC allowed excellent bone regeneration. The results suggest that BC can serve as both a barrier membrane and a sustained-release drug carrier, maintaining space and promoting bone re-formation. BC is an ideal biomaterial for dental implant surgery, facilitating sinus membrane elevation and exhibiting other clinical utilities.